Clinical and clinical research protocols in transplantation are increasingly employing potent lymphodepleting antibodies at the time of engraftment. Recently it has been appreciated that T cells in a lymphopenic environment undergo homeostatic expansion. Moreover, as a result of homeostatic proliferation, even previously naive T cells may acquire phenotypic and functional characteristics of memory cells, including the ability to respond to low concentrations of antigen without a need for CD28 costimulation. Using mouse systems which model the degree of lymphodepletion achieved clinically, we have found that residual non-depleted T cells undergo homeostatic expansion. We also find that homeostatic expansion induces long-lived resistance to tolerance induction by blockade of the CD28:B7 and/or CD154:CD40 pathways, neither of which are able to block homeostatic proliferation. Moreover, tolerance resistance is dominant (i.e., it can be adoptively transferred to naive mice). These findings are consistent with known behavior of memory T cells, and the known ability of memory cells to resist tolerance induction by blockade of CD28 and/or CD154. Our working model is that homeostatic expansion of T cells induces the development of at least a subset of memory-like cells, which subsequently interfere with tolerance induction. Our long-term goals are to identify this cell or cells and develop targeted approaches to enable tolerance. While tolerizing memory cells has traditionally proven quite difficult, recent data suggests that the "new" costimulatory receptors ICOS and OX40 (CD134) are extremely promising targets. In aim #1, we will define surface phenotype and costimulatory receptors used by "post-homeostatic" resistant cells. In particular, we hypothesize that both ICOS and CD134 will play an important role in activation, proliferation and survival of homeostatically proliferating T cells. Aim #2 will test the corollary hypothesis, that blockade of these pathways (and/or others identified in aim #1), along with B7 and/or CD154 may result in inhibition of homeostatic proliferation and overcome the resistance to transplantation tolerance mediated by homeostatically proliferating T cells. Collectively these studies will be important steps towards the development of approaches that complement lymphodepletion, and enable it to be used for tolerance induction. These approaches may also prove valuable in the setting of established T cell memory, such as may occur in sensitized recipients.